International Food Research Journal 16: 113-118 (2009)

Rapid isolation of genomic DNA from Asian green-lipped mussel

(Perna viridis) for random amplified microsatellite polymorphism
*Chai, L. C., 1Fatimah, C. A., 1Norhisyam, M. S., 1Rozila, A., 1Nadzirah, A.
S. and 2Natasha, L. H. Y.

1,2,

Faculty of Biotechnology and Life Sciences, Universiti Industri Selangor, 40000 Shah
Alam, Selangor, Malaysia
2
Center of Excellence for Food Safety Research, Faculty of Food Science and
Technology, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia

Abstract: The objective of the present study was to develop a rapid, reliable and yet inexpensive protocol for
genomic DNA extraction from frozen and ethanol-preserved Asian green-lipped mussels for random amplified
microsatelite (RAM) analysis. The procedure comprised of three major steps: (1) Tissue degradation by boiling
in 6% Chelex 100 resin in TE buffer; (2) Protein digestion by Proteinase K; and (3) DNA precipitation by adding
2 volumes of cold absolute ethanol. The entire procedure can be completed within two hours. The resulting
RAM profiles were clear and reproducible. Our results demonstrate that the combined protocol of Chelex 100Proteinase K-ethanol precipitation is a powerful yet economical DNA isolation method for population genetic
studies involving a large sample size.
Keywords: DNA extraction, mussel, Chelex 100 resin, RAM

Introduction
The Asian green-lipped mussel, Perna viridis
(L.) (Class Bivalvia: Family Mytilidae), is widely
distributed in the Indo-Pacific region (Siddall, 1980).
It can be found along the west and east coasts of
Peninsular Malaysia and also in certain areas of Sabah.
It is an economical source of animal protein for human
consumption. Today, it is being extensively cultured
in many Asian countries including Malaysia (Rosell,
1991; Monirith et al., 2003). Due to its widespread
distribution in this region, this species has been an
important bioindicator of a wide range of heavy metal
contaminants in the marine environments (Nicholson
and Lam, 2005; Lily et al., 2005). A number of studies
have been carried out to investigate the population
genetics and also in improving the broodstocks of this
commercially important seafood delicacy and hence
increase the productivity of mussel hatchery (Yap et
al., 2002; Yap et al., 2004; Lily et al., 2005).
Recently, polymerase chain reaction (PCR)
method has been widely applied in modern studies
of population genetics for detecting genetic diversity

*Corresponding author.
Email: layching.chai@gmail.com

within and among populations. However, the

feasibility of such studies is always limited by the
lengthy and labour-intensive procedure of DNA
isolation from the species. Realizing the need for a
rapid and simple procedure in DNA isolation from
marine species particularly in bivalve, a number of
studies have reported on the development of a rapid
procedure for DNA extraction (Banerjee et al., 1995;
Estoup et al., 1996; Nelson et al., 1998; Taris et al.,
2005; Aranishi and Okimoto, 2006). However, a
majority of the rapid methods were developed for
PCR detection of certain genes which were not
always suitable and applicable for molecular typing.
Here, we developed a rapid, simple and
inexpensive method for DNA isolation from
the mantle of Asian green-lipped mussel (Perna
viridis) for PCR amplification of random amplified
microsatellite (RAM). The DNA isolation procedure
is applicable for DNA extraction from frozen and
ethanol-preserved mussel, and is a suitable procedure
for phylogenetic studies involving a large number
of specimens in either frozen or ethanol-preserved
form.

All Right Reserved

114

Chai, L. C., Fatimah, C. A., Norhisyam, M. S., Rozila, A., Nadzirah, A. S. and Natasha, L. H. Y.

Materials and methods

Asian green-lipped mussels
Asian green-lipped mussels were collected
from a commercial mussel hatchery situated along
the west coast of Peninsular Malaysia. The mussels
were washed to remove the mud and the mantles
were separated from the mussels. Half portion of
the mantle tissue was packed into an individual bag
and was kept at -20oC; another half was immersed
in absolute ethanol and kept at 4oC. All processed
specimens were kept for several days before DNA
extraction.
DNA extraction
Approximately 25 mg of fresh or ethanolpreserved mussel mantle tissues were weighted into
sterile 2 ml microcentrifuge tubes containing 600
ul of Chelex 100 resin (Sigma) in TE buffer (10
mM Tris-HCl, pH 7.5; 0.1 mM of EDTA, pH8.0).
The tubes were boiled in a water bath for 10 min,
and then they were cooled down to 55oC. Thirty
microliters of 20 mg/ml Proteinase K (Sigma) was
added to each tube and followed by incubation at
55oC for 1 hour. The tubes were mixed by gently
flicking every 15 min. After that, the tubes were
again boiled for 5 min. Following centrifugation at
10 000 g for 5 min, the precipitate containing Chelex
100 resin and cell debris was discarded. One hundred

microliters of the supernatant containing DNA was

transferred into three individual 1.5 ml tubes each.
The first tube containing 180 l of DNA lysate was
immediately kept in 4oC for PCR amplification while
the other two proceeded with DNA purification
steps. A volume of 540 l of 6 M NaI (Sigma) and
10 l of 100% (wt/vol) silica (Yue and Orban, 2001)
were added into one of the tubes with 180 l of DNA
lysate. The tube was vortexed briefly then shaken
gently for 1 min and centrifuged at 10 000 g for 3-5
sec. The supernatant was discarded and the pellet
was washed with 1 ml of wash solution (10 mM Tris,
pH 7.5; 1 mM EDTA, pH 7.5; 100 mM NaCl, and
50% ethanol). The tube was centrifuged at 10 000 g
for 10 sec and the silica-bound genomic DNA was
dried at 37oC for 15 min. The DNA was then eluted
by adding 180 l of TE buffer and centrifuged at 10
000 g for 1 min. The supernatant containing genomic
DNA was transferred into new tubes. The remaining
tube with 180 l of supernatant proceeded with DNA
precipitation by absolute ethanol. A volume of 9 l of
5 M NaCl was added to the DNA lysate and followed
by 2 volumes of cold absolute ethanol. The mixture
was mixed by gently inverting the tube and then kept
at -20oC for 5 min. After that, centrifugation at 10
000 g was carried out for 5 min to pellet the genomic
DNA in the solution. The DNA pellet was dried at
37oC for 10 min and resuspended with 180 l of
sterile TE buffer.

Table 1. Purity (A260/A280) and Yield (ng) of genomic DNA obtained from frozen and ethanol-preserved
green-lipped mussels (Perna viridis) mantle by using various combinations of procedures
Chelex 100 resin
Proteinase K
Absolute ethanol
Silica
Puritya

Frozen

1.22 (0.09)

1.71 (0.08)

1.79 (0.01)

1.48 (0.16)

1.70 (0.08)

1.69 (0.20)

Ethanolpreserved

1.37 (0.05)

1.85 (0.08)

1.92 (0.04)

1.54 (0.12)

1.72 (0.02)

1.61 (0.09)

Yieldb

Frozen

45.30 (2.00)

13.70 (1.47)

8.67 (1.45)

6.47 (0.76)

5.43 (0.61)

11.10 (1.20)

(ng)

Ethanolpreserved

43.70 (1.30)

12.60 (1.60)

9.40 (0.56)

6.37 (1.29)

5.13 (0.23)

10.60 (1.31)

Purity of DNA is expressed in ratio of A260/A280

Total yield of DNA is calculated by using formula: (A260 - A320) x 50 (DNA extinction coefficient) x dilution
factor x final sample volume; and converted to nanogram scale by multiply by 1000
c
All data are recorded as: mean (standard deviation)
a

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Rapid isolation of genomic DNA from Asian green-lipped mussel (Perna viridis)

115

Figure 1. PCR amplification of random amplified microsatelite (RAM) of Asian green-lipped mussel using
genomic DNA prepared from: lane 1 to lane 3: Chelex 100-Proteinase K-ethanol precipitation protocol from
mussel A, B and C; lane 4 to lane 6: Chelex 100-Proteinase K-silica from mussel A, B and C; lane 7 to lane 8:
Chelex 100-ethanol precipitation from frozen and ethanol-preserved mussel. Lane M is 100-bp DNA ladder.
Mussel A, B, and C are three individual mussels preserved in ethanol

Quantitation of DNA
The quantity of DNA was determined by UV
spectrophotometry (Thermo). One hundred microliters
of DNA was added into tubes with 900 l of sterile
TE buffer to give a 1:10 dilution. Absorbance of the
DNA dilution was measured at 260nm, 280nm and
320nm. The yield and purity of DNA were calculated
as below:

30 sec at 94oC, 60 sec at 65oC and 30 sec at 72oC

for 35 cycles; and followed by a final extension of
5 min at 72oC. Amplification of PCR products were
fractionated by electrophoresis on 2% of agarose gel
and stained with ethidium bromide.

DNA yield (g) = (A260 - A320) x 50 (DNA extinction

In this study, different existing DNA extraction

protocols had been compared and a rapid, simple and
yet inexpensive protocol was developed by combining
and modifying existing protocols. The developed
protocol was tested on frozen and ethanol-preserved
mussels and the results obtained on DNA purity and
yield are summarised in Table 1. The protocol could
extract genomic DNA with a higher purity from
ethanol-preserved mussels (1.850.08) than from
frozen mussels (1.710.08) However, both frozen
and ethanol-preserved samples yielded comparable
amounts of DNA (13.701.47 ng and 12.601.60 ng
of DNA from frozen and ethanol-preserved mussels,
respectively). Although the A260/A280 ratio of DNA
from frozen mussel was lesser than 1.8, the quality
of the DNA extracted was still acceptable for PCR
amplification. Amplification of random amplified
microsatelite (RAM) by polymerase chain reaction

coefficient) x dilution factor x final sample volume

DNA purity = A260/A280
Random amplified microsatelite (RAM)
A 5-anchored oligonucleotide containing a
dinucloetide repeat of 5-NNN NNK KYW (BD)3
B(CA)10, in which K=(G or T), N=(A, C, G or T),
Y=(T or C), B=(C, G or T), D=(A, G or T), was
used for random amplified microsatellite analysis.
The PCR reaction mixture consisted of 25 ng of
genomic DNA, 1x PCR buffer, 1.5 mM MgCl2, 0.2
M primer, 100 M of dNTP mix and 1.5 U of Taq
DNA Polymerase (Promega), in a 10 l final volume.
PCR reactions were performed in a VeritiTM Thermal
Cycler (Applied Biosystem). The amplification
protocol was: 3 min at 94oC of pre-denaturation;

Results and discussion

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116

Chai, L. C., Fatimah, C. A., Norhisyam, M. S., Rozila, A., Nadzirah, A. S. and Natasha, L. H. Y.

Figure 2. PCR amplification of random amplified microsatelite (RAM) of Asian green-lipped mussel using genomic DNA prepared from: lane 1 and lane 2: Chelex 100-Proteinase K protocol from frozen and
ethanol-preserved mussel, respectively; lane 3 and lane 4: Proteinase K-ethanol precipitation protocol
from frozen and ethanol-preserved mussel, respectively. Lane M is 100-bp DNA ladder

(PCR) yielded distinct bands on gel electrophoresis

(Figure 1) and all the bands were reproducible.
The DNA extraction protocol was based on three
steps: (1) boiling the mantle tissue in 6% Chelex
100 resin; (2) Tissue digestion with Proteinase K;
and (3) DNA precipitation with absolute ethanol.
The whole protocol took only approximately 1
hours to complete and is suitable for DNA extraction
from a large number of samples. Previous studies
have reported successful PCR amplification using
standard Chelex-100 resin method (Chelex 100
combined with Proteinase K digestion) for DNA
isolation from ark shells and scallops (Steiner
and Muller, 1996), larvae and juveniles of oysters
(Launey and Hedgecock, 2001) and oyster parasites
(Ko et al., 1999). In our experiments, however,
DNA lysate obtained from Chelex 100-proteinase
K digestion method yielded only a few weak bands
in RAM analysis (Figure 2). Furthermore, the bands
were not reproducible and yielded different profiles
for each PCR amplification. UV spectrophotometry
revealed low purity in the DNA extraction (1.220.09
and 1.370.05 from frozen and ethanol-preserved
mussel, respectively) (Table 1). In fact, success in
PCR amplification depends heavily on the quality of
DNA template. Lower purity in DNA solution might
inhibit PCR amplification. However, under certain

circumstances, amplification by PCR can still take

place by using powerful Taq DNA polymerase
(Akalu and Ridchardt, 1999), optimization of PCR
condition, increasing the concentration of poorquality DNA (Fishback et al., 1999) and relief of
PCR inhibitory effect by BSA and T4 gene 32 protein
(Kreader, 1996). Poor-quality DNA might still be
applicable for PCR detection of specific genes, but
not suitable for molecular typing which requires
high quality DNA to ensure reproducibility of clear
multiple bands.
To explore the influence of Proteinase K and
Chelex 100 resin to the entire protocol, we performed
DNA extraction by omitting the use of Proteinase K
or Chelex 100 resin. The purity of DNA obtained
by both protocols was comparatively lower than the
DNA obtained by Chelex 100-Proteinase K-ethanol
precipitation protocol. Chelex 100 resin was proved
to play a role in increasing the purity of prepared
DNA. The protocol without usage of Chelex 100
resin yielded genomic DNA with A260/A280 ratio of
1.690.20 and 1.610.09 from frozen and ethanolpreserved mussel. Proteinase K, on the other hand,
was essential in protein digestion and releases more
DNA from the tissue. When the boiling in Chelex
100 resin was followed by ethanol precipitation, the
quantity of DNA extracted was significantly reduced

International Food Research Journal 16: 113-118

Rapid isolation of genomic DNA from Asian green-lipped mussel (Perna viridis)

as compared to the full protocol (Table 1). The

finding was in total agreement with the observation
by Yue and Orban (2001).
The DNA extraction protocol combining Chelex
100 and proteinase K digestion, followed by silica
purification was adapted and slightly modified from
the protocol designed for DNA extraction from fish
scales (Yue and Orban, 2001). The authors claimed
that their method was rapid and yielded high quality
DNA of approximately 50 to 700 ng per scale. In
the present experiment, the method yielded only
8.671.45 ng and 9.400.56 ng of DNA from
frozen and ethanol-preserved mussels, respectively.
Furthermore, although higher purity was obtained
by this method, it was comparatively more lengthy
and expensive than Chelex 100-proteinase K-ethanol
precipitation protocol. RAM profiles generated
with DNA extracted by Yue and Orbans protocol
were comparative with the profiles generated
with our protocol. However, our findings were
different from the outcomes reported by Aranishi
and Okimoto (2006). The workers suggested that
ethanol precipitation is unable to completely remove
the PCR-inhibiting materials present in the mantle
tissue that is rich in polysaccharide (Neudecker and
Grimm, 2000) after failure in PCR amplification
using the combined protocol of urea, Chelex 100
and proteinase K tissue digestion and followed by
ethanol precipitation of DNA in the experiment.
We speculated that the differences might be due to
the use of urea and other chemical detergents in the
digestion step that were not completely removed
from the genomic DNA by ethanol precipitation and
later inhibited the PCR amplification. In our protocol,
the lysis step was merely performed by proteinase K
in TE buffer and Chelex 100 resin. Thus, chemical
contamination of final DNA product was not an issue
in our procedure.
Our results demonstrate that the combined
protocol of Chelex 100-Proteinase K-ethanol
precipitation is a powerful yet economical method
for DNA isolation from frozen and ethanol-preserved
mussels for population phylogenetic studies. The
procedure uses no hazardous chemical, as well
as detergent in the DNA extraction and the full
procedure takes not more than 2 hours to complete.
Therefore, the protocol is suitable for investigating
a large number of mussels for population genetic
studies.

117

Acknowledgements
This work (Project No: 02-02-SF0008) was
supported by a grant from the Science Fund from
the Ministry of Science, Technology and Innovation,
Malaysia. The authors would like to thank Professor
Dr. Son Radu, Center of Excellence for Food Safety
Research, Faculty of Food Science and Technology,
Universiti Putra Malaysia for the research facilities.
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